A serum-free medium suitable for maintaining cell morphology and liver-specific function in induced human hepatocytes

Abstract

hiHep is a new type of hepatocyte-like cell that is predicted to be a potential unlimited source of hepatocytes for a bioartificial liver. However, hiHep cannot currently be used in clinical settings because serum must be added during the culture process. Thus, a defined medium is required. Because serum is complex, an efficient statistical approach based on the Plackett-Burman design was used. In this manner, an original medium and several significant cell growth factors were identified. These factors include insulin, V_H, and V_E, and the original medium was optimized based on these significant factors. Additionally, hiHep liver-specific functions and metabolism in the optimized serum-free medium were measured. Results showed that hiHep functions, such as glycogen storage, albumin secretion, and urea production, were well maintained in our optimized serum-free medium. In summary, we created a chemically defined, serum-free medium in which cell growth, proliferation, metabolism, and function were well maintained. This medium has the potential to support the clinical use of hiHep.

Keywords: Bioartificial liver; Medium; Serum-free; hiHep.

Fig. 1

The effect of factors in the medium. Normal plot of the standardized effects of cell growth factors in a Plackett–Burman design (The significance level of each variable effect was determined using t distribution associated with Student’s t-test,; The red vertical line means the t critical value (α = 0.1). (Color figure online)

Fig. 2

Screening of the original medium. hiHep was seeded in 24-well plates and cultured in the indicated medium for 8 days. After culturing, cells were subjected to CCK-8 analysis to evaluate their growth. Cells cultured in HMM served as the control

Fig. 3

The effect of significant factors on cell growth. a The growth of hiHep cells in different media that were supplemented with respective factors. 1: ORM + insulin, 2: ORM + V_E, 3: ORM + V_H, 4: ORM + V_E + V_H + insulin (medium 4 was called the optimized medium). Data are present as the mean ± SD of six independent experiments. *p < 0.05 versus ORM

Fig. 4

Cell growth results. A hiHep morphology in ORM, OPM and HMM; (a) ORM; (b)OPM; (c) HMM. B The hiHep growth curves

Fig. 5

Characterization of hiHep metabolism and damage. a Glucose concentration; b Lactate production; c relative consumption of eight essential amino acids (The variation of amino acids concentration in OPM versus the corresponding of HMM); d LDH activity; e Transaminase activity. Data are presented as the mean ± SD of three independent experiments. *p < 0.05

Fig. 6

Assessment of hiHep cell functionality. A Liver-specific gene analysis by semi-quantitative PCR; B Cells on day 8 showed the capability to store glycogen, as seen in the areas stained in pink. (a) OPM, (b) HMM; C Albumin secretion during the culture process; D Urea production during the culture process; E CYP1A2 activity; F CYP3A4 activity (The activity of CYP was reflected by the reduction of corresponding substrate concentration, the greater variation means the higher activity). *p < 0.05 versus HMM

Fig. 7

Long-term cell culture. a hiHep cell morphology in each passage (100 μΜ); b hiHep cell proliferation in each passage; c mRNA expression levels in liver-specific functions (i.e., ALB, AAT, TAT, CDH1, C5and TTR) in hiHep cells as tested by qRT-PCR. The data are expressed as the mean ± SD (n = 3). *p < 0.05 versus HMM

Fig. 7

Long-term cell culture. a hiHep cell morphology in each passage (100 μΜ); b hiHep cell proliferation in each passage; c mRNA expression levels in liver-specific functions (i.e., ALB, AAT, TAT, CDH1, C5and TTR) in hiHep cells as tested by qRT-PCR. The data are expressed as the mean ± SD (n = 3). *p < 0.05 versus HMM